CF is an autosomal recessive genetic disorder affecting approximately 30,000 patients in the United States and 60,000 patients worldwide, with an average life expectancy of about 30 years. Lung infection with Pseudomonas aeruginosa is a leading cause of mortality in cystic fibrosis (CF) patients. The bacteria establish biofilms through the production of an exopolysaccharide called alginate. Growth in biofilms protects the organism from antibiotics and host defense mechanisms. Generally these chronic infections are not eradicated despite aggressive antibiotic therapy, thus new treatments are needed. This proposal will develop a novel therapy that will disrupt the biofilm and kill the bacteria. Alginate lyase (AL) will be chemically linked to the bactericidal peptide, CAP18. Digestion of the alginate by this conjugate will deliver the cytotoxic CAP18 peptide to the bacteria, as well as increase its susceptibility to antibiotics and host defense mechanisms. Furthermore, since CAP18 peptides can bind and neutralize lipopolysaccharide (LPS), an additional benefit will be inhibition of inflammation which contributes to loss of pulmonary function in CF patients. This proposal will clone, express, and purify recombinant AL which will then be chemically linked to a potently bactericidal synthetic peptide derived from CAP18. The conjugate, AL-CAP18 will be tested in several in vitro assays to verify the fusion still retains the functional activity of each component. This will include neutralization of LPS, bactericidal activity, AL enzyme activity, promotion of antibiotic diffusion through alginate, and promotion of neutrophil phagocytosis and killing of P. aeruginosa. The ability of AL-CAP18 to function in the presence of sputum from CF patients will be evaluated with respect to reduction of viscosity and bactericidal effects. The potential of AL-CAP18 to augment or synergize with conventional antibiotics and recombinant DNase will be investigated. This novel therapy will provide a bactericidal agent that can kill antibiotic-resistant bacteria combined with an enzyme that will disrupt the architecture of the biofilm. This treatment should potentiate the penetration of conventional antibiotics and natural host defense mechanisms into the biofilm to thus gain access to the bacteria. Currently, there is no approved drug that can dissolve bacterial alginate, thus AL-CAP18 represents a new therapy with a unique mechanism of action to benefit CF patients.